Variola (smallpox) remains a bioterrorism threat in the 21st century. The Dryvax vaccine is too toxic for many persons. Little is known about the specific T cell response induced by intradermal Dryvax. We propose detailed studies of the epitopes and antigens recognized by vaccinia-specific CD8 and CD4 T-cells in humans. The planned antigen discovery method, expression cloning, interrogates the viral proteome with libraries of cloned vaccinia genomic DNA. We have successfully used this method with HSV-2, which has a complex genome similar to that of vaccinia. The responses of vaccinia-specific human T-cells lines and clones will be validated in quantitative measurements of immune responses using direct ex vivo assays. In Aim 1, we will derive CDS CTL lines and clones that are specific for vaccinia, and determine which vaccinia open reading frames and peptide epitopes they recognize. Epitopes will be validated in both CTL and IFN- gamma assays and dose-response curves done to document high-avidity recognition. We will then measure the diversity of the CDS response and the evolution of the response over a one year period in a subset of primary vaccinees. We predict that the immunodominant CDS antigens will be non-structural early proteins, and that immunodominance will also be controlled by HLA type and will be stable over time. CD4 T-cells provide important help for antibody and CDS responses. Therefore, in Aim 2, we will detemine the fine specificity of vaccinia-specific CD4 T-cell lines and clones using our proven molecular library method. Selected vaccinia proteins that stimulate CD4 T-cell responses will be expressed as full-length proteins in baculovirus to study CD4 T-cell immunodominance in a population of vaccinated subjects. We predict that structural proteins will be dominant and that the CD4 response will include reactivity with vaccinia proteins that are the targets of neutralizing antibodies. Several such proteins are already available as baculovirus constructs. The data will be medically useful in evaluating the immunogenicity of candidate smallpox vaccines that are being evaluated for widespread use in the case of possible variola exposure. Poxviruses expressing heterologous microbial or cancer proteins are being modified for increased immunogenicity, with the goal of prevention and therapy of other infections and malignancies. Our data will assist in evaluating the immunogenicity of these constructs. [unreadable] [unreadable] [unreadable]